Correlation between immune‐related Tryptophan‐Kynurenine pathway and severity of severe pneumonia and inflammation‐related polyunsaturated fatty acids

Abstract Background Immune dysfunction and oxidative stress caused by severe pneumonia can lead to multiple organ dysfunction and even death, causing a significant impact on health and the economy. Currently, great progress has been made in the diagnosis and treatment of this disease, but the mortality rate remains high (approximately 50%). Therefore, there is still potential for further exploration of the immune response mechanisms against severe pneumonia. Objective This study analyzed the difference in serum metabolic profiles between patients with severe pneumonia and health individuals through metabolomics, aiming to uncover the correlation between the Tryptophan‐Kynurenine pathway and the severity of severe pneumonia, as well as N‐3/N‐6 polyunsaturated fatty acids (PUFAs). Methods In this study, 44 patients with severe pneumonia and 37 health controls were selected. According to the changes in the disease symptoms within the 7 days of admission, the patients were divided into aggravation (n = 22) and remission (n = 22) groups. Targeted metabolomics techniques were performed to quantify serum metabolites and analyze changes between groups. Results Metabolomics analysis showed that serum kynurenine and kynurenine/tryptophan (K/T) were significantly increased and tryptophan was significantly decreased in patients with severe pneumonia; HETE and HEPE in lipids increased significantly, while eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), α‐linolenic acid (linolenic acid, α‐LNA), arachidonic acid (ARA), Dihomo‐γ‐linolenic acid (DGLA), and 13(s)‐hydroperoxylinoleic acid (HPODE) decreased significantly. Additionally, the longitudinal comparison revealed that Linolenic acid, DPA, and Tryptophan increased significantly in the remission group, while and kynurenine and K/T decreased significantly. In the aggravation group, Kynurenine and K/T increased significantly, while ARA, 8(S)‐hydroxyeicosatetraenoic acid (HETE), 11(S)‐HETE, and Tryptophan decreased significantly. The correlation analysis matrix demonstrated that Tryptophan was positively correlated with DGLA, 12(S)‐hydroxyeicosapentaenoic acid (HEPE), ARA, EPA, α‐LNA, DHA, and DPA. Kynurenine was positively correlated with 8(S)‐HETE and negatively correlated with DHA. Additionally, K/T was negatively correlated with DGLA, ARA, EPA, α‐LNA, DHA, and DPA. Conclusion This study revealed that during severe pneumonia, the Tryptophan‐Kynurenine pathway was activated and was positively correlated with the disease progression. On the other hand, the activation of the Tryptophan‐Kynurenine pathway was negatively correlated with N‐3/N‐6 PUFAs.


| INTRODUCTION
6][7] Indoleamine 2,3dioxygenase (IDO) is a key enzyme in the Tryptophan-Kynurenine pathway and can be considered a marker of interferon γ (IFN-γ)-mediated immune activation, the enzyme's activity can usually be expressed by the ratio of kynurenine to tryptophan (K/T).It plays an important role in inducing immune tolerance by creating a low tryptophan environment and generating metabolites such as kynurenine in local tissues to mediate immunosuppressive effects in the body. 8-3/N-6 PUFAs are important indicators of the severity of inflammation.N-3 PUFAs have antiinflammatory effects by reducing the production of inflammatory factors, thereby decreasing the inflammatory response in lung tissue.In contrast, high levels of N-6 PUFAs can increase the production of inflammatory factors and may worsen the symptoms and severity of pneumonia.Furthermore, the former can attenuate the pro-inflammatory effects of the latter.[9][10][11][12] Existing studies have found a negative correlation between the intake of N-3 PUFAs and IDO activity, which may indicate a reduction in immune activation when the intake of N-3 PUFAs is higher.13 At the same time, N-3 PUFAs can inhibit LPS-induced inflammationassociated depression by suppressing IDO expression.14 Furthermore, metabolites of EPA can inhibit the expression of IDO in dendritic cells (DCs) and tumor cells.15,16 Nesrine Kamal Bassal et al. have demonstrated that ARA can inhibit the activity of IDO in human monocytes.17 This indicates that N-3/N-6 PUFAs have a regulatory effect on IDO activity and may influence the development of certain diseases.Therefore, we aim to investigate the changes and correlations between N-3/N-6 PUFAs and the Tryptophan-Kynurenine pathway during severe pneumonia.This research is expected to provide additional theoretical basis for the immune regulatory mechanisms in severe pneumonia.Exclusion criteria: age ≤18 years; end-stage renal disease, end-stage liver disease, diabetes, active pulmonary tuberculosis, lung transplantation, noninfectious interstitial lung disease, pulmonary embolism, severe immunosuppression, malignant tumor, pregnancy, hemodialysis, blood transfusion, and plasma exchange patients.
Serum samples and clinical data were collected at admission and 1 week after admission.Patients were divided into exacerbation and remission groups based on their condition at the two time points.Evaluation was performed by three clinicians and two researchers based on the patient's examination results or symptoms, including pneumonia severity index (PSI) score, pulmonary imaging lesion range, inflammatory markers, arterial blood gas, and so forth.

| Sample collection and preparation
Collection: Each patient at least two times during hospitalization, each time take 5 mL venous blood, all in the morning and overnight eating state, stood for 5 min after centrifugation to collect serum supernatant and stored in −80℃ refrigerator.
Preparation: Preparation of serum samples is divided into extraction and derivatization, serum samples thawed at 4℃ Take A, B two groups of EP tube, group A added 1 ppm Prostaglandin D2 (PGD2)-d4 5 μL, group B added HETE-d8 5 μL, take 50 uL serum into group A EP tube, then add 600 μL precooled methanol extraction, point shock 10-15 times fully mixed.The mixture was centrifuged at 13000 rpm for 5 min at 4℃, and the supernatant was transferred to the EP tube of group B for nitrogen blowing for about 30 min to obtain pale yellow crystals.Each 5 μL of 1-Hydroxybenzotriazole (HOBt), 5-(Diisopropylamino) Amylamine (DIAAA), and O-(7azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added in turn, and each addition interval required 30-60 s of oscillation.After that, it was diluted to 50 uL with acetonitrile, centrifuged at 13,000 rpm for 5 min at 4℃, and finally transfer 45 μL into the intubation for detection.
Mass spectrometry was conducted on an Agilent 6550 UHD accurate mass Q-TOF/MS system with a dual jet stream electrospray ion source (dual AJS ESI).The instrument was operated in positive full scan mode with the following MS parameters: dry gas temperature, 250℃; dry gas flow, 15 L/min; sheath gas temperature, 300℃; sheath gas flow, 11 L/min; nebulizer pressure, 20 psi; capillary voltage, 5000 V; and nozzle voltage, 500 V. Mass spectra were recorded between 200 and 1000 m/z.Accurate mass measurements were obtained by using a low flow of TOF reference mixture (reference masses: m/z 322.0481, 622.0289, 922.0098), containing internal reference masses at m/z 922.0098 (C18H18F24N3O6P3).

| Data analysis
Continuous variables in this study were expressed using the median (interquartile range) and categorical variables were expressed as numbers (frequency).Differences in continuous variables were tested using the Mann-Whitney-Wilcoxon rank-sum test (two groups) or the Kruskal-Wallis test (three or more groups).
Categorical data were compared between different groups using the Chi-square test (two groups) or Fisher's exact test (three or more groups).Correlation analysis was performed using Spearman's correlation coefficient with R package "corrplot."The significance level for the test was set to .05.Metabolite analysis was conducted using partial least squares discriminant analysis (PLS-DA) with R package "mixOmics."Criteria for screening significantly different metabolites were as follows:

| Characteristics of participants
Several parameters, including procalcitonin (PCT), pro brain natriuretic peptide (PRO-BNP), and D-Dimer, were measured in patients with severe pneumonia and health individuals.The results demonstrated that the patients with severe pneumonia exhibited higher values than the normal range.Furthermore, some parameters in the blood, neutrophil% (NEU%), neutrophil (NEU), and monocyte (MONO), were higher in patients with severe pneumonia than in the health control group (all p < .01).On the other hand, the values detected for lymphocyte% (LYM%), eosinophil% (EOS%), basophil (BAS%), lymphocyte (LYM), and hematocrit (HCT) parameters were lower than those in the health control group (all p < .01)(Table 1).

| Metabolic pathway construction and metabolite comparison
A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 272 metabolites.PLS-DA results showed that the severe pneumonia group was significantly separated from the health control group (Figure 1A).Additionally, good predictive ability and good fitting (R2Y = 0.604, Q2 = 0.572, p < .05)were detected by this model.Moreover, a volcano plot performed using serum of patients with severe pneumonia and health individuals revealed that 167 metabolites were downregulated (blue) while 14 metabolites were upregulated (red) compared to the health control group (Figure 1B).We used a heatmap to visualize the Tryptophan-Kynurenine pathway and the N-3/N-6 polyunsaturated fatty acids (Figure 1C).
To perform a longitudinal comparison, the patients with severe pneumonia were divided into two groups: the aggravation group and the remission group, according to the PSI score, arterial blood gas and other indicators during the disease progression.Furthermore, in this experiment, the differences in serum metabolites between the two groups were evaluated at two-time points, at admission and after 1 week of admission.The results demonstrated that Linolenic acid, DPA, and tryptophan increased significantly in the remission group (p = .029,0.041, and 0.035, respectively), but the kynurenine and K/ T decreased significantly in the same group (p = .004and <.001, respectively).On the other hand, the results showed a significant increase in the aggravation group's Kynurenine and K/T (p = .039and <.001, respectively).However, in this group, a significant decrease was detected for ARA, 8(S)-HETE, 11(S)-HETE, and tryptophan (p = .014,.015,.041,and .021,respectively) (Figure 4).The detailed parameters are reported in Supporting Information Table   In this study, the relationships between Tryptophan-Kynurenine pathway metabolites and N-3/N-6 PUFAs metabolites were investigated by Spearman's rank correlation coefficient (Figure 5)，The detailed values of correlation coefficients and P-values were shown in Supporting Information Table 2.The results demonstrated that Tryptophan was positively correlated with DGLA, 12(S)-HEPE, ARA, EPA, α-LNA, DHA, and DPA (r = .50,.25,.71,.62,.69,.66,and .72,respectively, p < .05),and kynurenine was positively correlated with 8(S)-HETE (r = .24,p = .038)and negatively correlated with DHA (r = −.35,p < .01).Additionally, K/T was negatively correlated with DGLA, ARA, EPA, α-LNA, DHA, and DPA (r = −.37,−.65, −.62, −.62, −.71, and −.68, respectively, p < .01).

| DISCUSSION
Metabolites, derived from human pathophysiological activities, can reflect the state of the body.Therefore, it has been described that metabolomics has great potential for diagnosis and disease prediction.Furthermore, this tool can be applied to explore biochemical mechanisms associated with conditions.Therefore, targeted metabolomics was used in this study to identify significant alterations in metabolic pathways in patients with severe pneumonia, focusing on the link between the Tryptophan-Kynurenine pathway, N-3/N-6 PUFAs metabolism, and severe pneumonia.

| Tryptophan-Kynurenine pathway regulates the body's immune function
According to the results, the Tryptophan-Kynurenine pathway was one of the metabolic pathways significantly affected in severe pneumonia.The cross-sectional study demonstrated that in patients with severe pneumonia, there was a significant decrease in tryptophan, hydroxykynurenine, and AMDA, along with a notable increase in kynurenine and K/T.Furthermore, through longitudinal comparison, it was observed that tryptophan exhibited a significant increase with disease recovery, whereas kynurenine and K/T exhibited a significant decrease.Conversely, with disease progression, kynurenine and K/T showed a significant increase, while tryptophan exhibited a substantial reduction.In addition, it has been described that Tryptophan-Kynurenine pathway activation could mediate immunosuppressive function. 18DO is a crucial enzyme in the Tryptophan-Kynurenine pathway, and an innate immune inhibitor produced endogenously by epithelial and dendritic cells. 19The enzyme's activity can usually be expressed by the ratio of K/T. 20When dendritic cells are stimulated by pro-inflammatory factors, including IFN-γ, tumor necrosis factor α, and interleukin-6, the activity of IDO increases, leading to the accumulation of kynurenine. 21,22This, through the aryl hydrocarbon receptor pathway, diminishes the ability of lung epithelial cells to produce immune mediators and suppresses the activity of CD4 + T cells, thereby preventing Th cell-mediated immune injury induced by acute infections. 23On the other hand, the heightened activity of IDO can trigger autophagy in epithelial cells, exerting a negative impact on the secretion of inflammatory cytokines. 24In addition, the IDO activity increases can also inhibit T cell proliferation and activation by reducing the energy source. 25,26ydroxykynurenine, 3-hydroxy-anthranilic acid, and kynurenic acid are downstream derivatives of kynurenine, and the rate-limiting enzymes are Kynurenine-3monooxygenase, kynureninase, and kynurenine aminotransferase.It has been described that hydroxykynurenine and 3-hydroxy-anthranilic acid are converted from a reduced state to strong oxidants in the presence of molecular oxygen, inducing oxidative stress, injury, and apoptosis of various cells 27 and inducing lung injury in some inflammatory diseases. 28Additionally, AMDA is an intermediate between kynurenine and kynurenic acid, which can be spontaneously converted into kynurenic acid, determining the levels of kynurenic acid in the organism.Therefore, in this study, AMDA was used to evaluate the kynurenic acid levels in patients with severe pneumonia.][31][32] Therefore, this study hypothesized that the Tryptophan-Kynurenine pathway is activated in severe pneumonia to prevent self-damage caused by excessive immune activation.The degree of pathway activation is directly proportional to the severity of the disease.
4.2 | N-3/N-6 polyunsaturated fatty acid metabolism and inflammatory immunity Changes in lipid metabolism are closely related to inflammatory immunity, energy metabolism, tissue repair, and biosynthesis in pathological conditions.It has been suggested that N-3 and N-6 PUFAs have antiinflammatory and pro-inflammatory effects, respectively.N-3 PUFAs and their metabolites can promote the formation of biologically less active leukotriene B5, and inhibit the cytokines production by inflammatory cells, playing a biological activity in the inflammatory regression period.4][35] On the other hand, prostaglandins and leukotrienes can be synthesized by N-6 PUFAs and promote leukocyte chemotaxis, produce inflammatory factors, and improve vascular permeability.However, some reports demonstrated that an increased intake of N-6 PUFAs did not impact inflammation. 9We hypothesized that the significant decrease of N-3/N-6 PUFAs in this study may be due to the large amount of consumption during pathological reactions, which exerts pro-inflammatory and anti-inflammatory properties.
Another crucial factor, HETEs, was also evaluated in this study.HETEs, downstream derivatives of arachidonic acid produced by lipoxygenase (LOX) and glutathione peroxidase (GPX) enzymes, are positively correlated with the inflammatory response.This biological pathway can be activated when respiratory epithelial cells are damaged or infected by pathogens.Some subtypes of LOX enzymes can induce inflammation by acting on different sites producing 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE.In addition, these HETEs can accumulate and activate peroxisome proliferatoractivated receptors (PPARs) to enhance the inflammatory response.][38][39] The substrates of N-3 PUFAs (such as α-linolenic acid) and N-6 PUFAs (including linoleic acid, γ-linolenic acid, and ARA) are phosphatidylcholines, and it has been described that the activation of these two pathways could inhibit each other. 40However, our research findings indicate that levels of N-3 fatty acid metabolites EPA, DPA, DHA, α-LNA, and N-6 fatty acid metabolites ARA were simultaneously reduced in patients with severe pneumonia, while levels of HETEs and HEPEs were elevated.This suggests that under a steadystate regulatory mechanism, these two pathways exhibit antagonistic and synergistic effects.

| Correlation analysis between Tryptophan-Kynurenine pathway and inflammation-related polyunsaturated fatty acid metabolism
In this study, we found that severe pneumonia activated the Tryptophan-Kynurenine pathway and N-3/N-6 PUFAs metabolism，while the results obtained from the correlation matrix analysis demonstrated that the Tryptophan-Kynurenine pathway activation was positively correlated with the metabolism of N-3/N-6 PUFAs.Some studies demonstrated that EPA could inhibit IDO expression in tumor cells, 15 while EPA-derived metabolite resolvin E1 can induce several biological responses, including inhibition of dendritic cell migration and leukocyte infiltration, modulation of IDO synthesis in dendritic cells, inhibition of T cell activation, and modulation of effector T cell death. 16Furthermore, AA, DHA and dihomo-γ-linolenic acid are potent inhibitors of IDO in human acute monocytic leukemic THP-1 cells and primary human monocytes. 17,41In addition, some reports revealed that an increase in activity of IDO can promote fatty acid oxidation. 42Recently, Ele and collaborators found that IDO can promote the downregulation of essential enzymes related to fatty acid synthesis in CD4 + T cells, inhibit fatty acid synthesis, and CD4 + T cell proliferation and differentiation. 43herefore, we hypothesized that severe pneumonia accelerates the metabolism of N-3/N-6 PUFAs and relieves Inhibition of IDO activity, thereby enhancing immunosuppression effect and reducing autoimmune destruction.
However, our study has some limitations.First, it is a small-scale, single-center study, and mechanical ventilation and medication interventions in some patients may affect the changes in certain metabolites.Second, the study only demonstrates the correlation between the immune-related Tryptophan-Kynurenine pathway, the severity of severe pneumonia, and the inflammation-related PUFAs, without capturing the interactions between these factors, which would require extensive intervention studies for validation.Lastly, since severe pneumonia is often caused by a mixed infection of multiple pathogens, this study did not differentiate between pathogens.However, it has been found that various bacterial, fungal, and viral infections can induce the expression of IDO, [44][45][46][47][48][49][50][51][52][53] and tryptophan can serve as a prognostic biomarker for COVID-19, but future research still needs more detailed classification of the pathogen. 6,54

| CONCLUSION
In this study, metabolomics analysis was conducted on the serum of patients with severe pneumonia and health individuals.The results showed that the Tryptophan-Kynurenine pathway was activated in severe pneumonia and positively correlated with disease severity, reducing self-immune damage and oxidative stress by suppressing immune reactions.In contrast, the severity of the disease was negatively correlated with N-3/N-6 PUFAs.Therefore, we conclude that the activation of the Tryptophan-Kynurenine pathway in patients with severe pneumonia is inversely correlated with N-3/N-6 PUFAs.Thus, a relationship between inflammation, the Tryptophan-Kynurenine pathway, and N-3/N-6 PUFAs metabolism was established to evaluate disease severity.This study may also be relevant to the development of precision interventions for the progression and treatment of severe pneumonia.The mechanism by which N-3/N-6 PUFAs affect immune regulation in severe pneumonia by regulating the Tryptophan-Kynurenine pathway is worthy of further exploration.

2 | METHOD 2 . 1 |
Patients and control group A total of 81 subjects were included in the study, including 44 patients with severe pneumonia in the Department of Respiratory Medicine of the First Affiliated Hospital of Guangzhou Medical University, and the remaining 37 health controls.Participants or their families signed informed consent.This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.Inclusion criteria: age ≥18 years; refer to the American Thoracic Society and Infectious Diseases Society of America severe pneumonia standard, meet one of the following major criteria or ≥3 minor criteria.Main criteria: (1) Intubation requires mechanical ventilation; (2) Vascular active drugs are still needed after active fluid resuscitation of septic shock.Secondary criteria: (1) Respiratory frequency ≥30 times/min; (2) Pa02/Fi02 ≤ 250 mmHg; (3) Multilobe infiltration; (4) Disorders of consciousness and/or orientation; (5) Blood urea nitrogen ≥20 mg/dL; (6) Leukopenia (white blood cell [WBC] < 4×10 9 /L); (7) Thrombocytopenia (platelet [PLT] < 100 × 10 9 /L); (8) Hypothermic (central body temperature <36℃); (9) Hypotension requires fluid resuscitation.

F
I G U R E 1 Metabolic analysis of patients with severe pneumonia.(A) Partial least squares discriminant analysis (PLS-DA) analysis showed a significant separation of serum metabolic phenotypes between patients with severe pneumonia and health people.(B) Volcano plot of targeted metabolomics analysis showed top serum metabolites that increased (red) or decreased (blue) in severe pneumonia compared to controls.(C) Heatmap analysis emphasized the significant changes in N-3/N-6 polyunsaturated fatty acids and the kynurenine pathway in patients with severe pneumonia compared with health people.F I G U R E 2 Tryptophan-Kynurenine metabolic pathway was significantly affected by severe pneumonia, and the asterisk indicated the significance.(*p <.05; **p < .01;***p < .001).F I G U R E 3 (A) The N-6 fatty acid metabolic pathway.(B) The N-3 fatty acid metabolic pathway.The asterisk indicates the significance.(*p < .05;**p < .01;***p < .001).

F I G U R E 4
Longitudinal time-point comparison of severe pneumonia patients at various disease stages.
Characteristics of included patients and health individuals.
T A B L E 1